Plasminogen activators have received attention for their role in the fibrinolytic system. These enzymes catalyze the conversion of the proenzyme plasminogen into the proteolytic enzyme plasmin; plasmin can, in turn, degrade fibrin, a major component of blood clots. Thus, plasminogen activators are potentially useful for the therapeutic treatment of blood clots.
The known plasminogen activators include streptokinase, which is of bacterial origin, urokinase (u-PA), which has been isolated from urine and culture fluids, and tissue plasminogen activator (t-PA), which is available from cultured human cells (Rifkin et al., J. Exp. Med. 139:1317-1328 (1974); Wilson et al, Cancer Res. 40:933-938 (1980)). Streptokinase and u-PA are available commercially, but appear not to possess the therapeutic efficacy of t-PA.
Intact t-PA is a glycoprotein having a molecular weight of about 66,000 daltons, and exists as either a one-chain polypeptide (Binder et al., J. Biol. Chem. 254:1998-2003 (1979)) or it may be cleaved by plasmin (Wallen et al., Prog. in Fibrinolysis 5:16-23, (1981)), into a two-chain form, wherein the two polypeptides are linked by a disulfide bond (Rijken et al., Biochem. Biophys. Acta 580:140-153 (1979)). Non-glycosylated, enzymatically active t-PA has been produced in eukaryotic cells grown in the presence of drugs that prevent glycosylation (Little et al., Biochemistry 23:6191-6196 1984)); and in bacteria (Pennica et al., Nature (London) 301:214-221 (1983)). Degraded forms of t-PA, having molecular weights of approximately 50,000 and 32,000 have been found coexisting with intact, one-chain and two-chain t-PA (Granelli - Piperino & Reich, J. Exp. Med. 148:223-234 (1978)). Prior art methods for isolating t-PA have not been particularly effective at separating the degraded forms of t-PA from the intact t-PA.
In pharmaceutical formulations of t-PA, the availability of substantial quantities of pure intact single-chain enzyme is important and desired. The strong fibrin binding exhibited by t-PA (Thorsen et al., Throm. Diath. Haemorrh. 28:65-74 (1972)) is believed to be important for its therapeutic efficacy. The lower molecular weight degraded forms, which have aberrant fibrin binding properties (Banyai et al., FEBS Lett. 163:37-41 (1983)), do not appear to display the specificity and clot localization properties of intact one-chain and two-chain t-PA. Further, it is believed that single-chain t-PA is more desirable in pharmaceutical formulation than the two-chain form due to the much slower rate at which the single-chain form is inactivated by specific inhibitors of t-PA found in plasma (Lecander et al., Brit. J. Haematol. 57:407-412 (1984)) and due to the potential systemic activation caused by two-chain t-PA. In addition commercially available preparations of t-PA are limited to administration at concentrations at or below 1 mg/ml. For initial administration of t-PA, higher concentrations of t-PA would be preferable.
Various protocols have been described for the purification of t-PA using chromatographic, electrophoretic, and selective extraction and precipitation methods. Most of these methods, including a widely used purification (Rijken and Collen, J. Biol. Chem. 256:7035-7041 (1981)), are not appropriate for the large scale production of t-PA as they are inefficient in product recovery, only partially effective in removing impurities, or use adsorbents which may introduce toxic, mitogenic, tumorogenic or immunogenic ligands into the t-PA preparation (Reagan et al., Throm. Research 40:1-9 1985)). Large scale purification methods employing immunoaffinity chromatography (Wallen et al., Eur. J. Biochem. 132:681-686 1983); Nielsen et al. EMBO J. 2:115-119 (1983)) are limited by the cost of the antibody resin, the difficulty in sterilizing or sanitizing this resin and by the potential for the antibody or fragments of the antibody leaching into the recovered t-PA. In addition, the published methods do not provide procedures to concentrate t-PA to give useful therapeutic formulations. Furthermore, the presence of degraded forms of t-PA in preparations of the purified enzyme remains problematic to those skilled in the art (Kruithof et al., Biochem. J. 226:631-636 (1985)). Deqraded t-PA is commonly found in fermentation broth. Degraded t-PA not only dilutes the intact t-PA, but in addition, as mentioned above, it is not specific and is less able to localize clots as the intact t-PA. Therefore, contamination of final t-PA product with degraded t-PA provides serious drawbacks to the product as a therapeutic agent. Large scale chromatographic methods for the specific recovery of intact t-PA free from degraded forms have not been reported. The method disclosed by Rijken and Collen, supra, fails to separate intact t-PA from its degraded forms, and the two forms have consistently co- purified together.
Most tissue culture cells require serum supplementation of media for optimal growth and survival. The known methods for recovery of t-PA from conditioned tissue culture media are generally effective only when serum-free media is used. In those examples wherein serum containing production medium is used (Reagen et al., supra: Cederholm-Williams & Porter, Brit. J. Dermatology 110:423-429 (1984), Kluft et al., Adv. Biotechnol. Processes 2:97-110 (1983)) only partially pure t-PA or t-PA containing degradation products were primarily recovered. This degradation is attributed to serum components and may be only partially blocked by the addition of proteinase inhibitors (Reagen et al., supra). Thus it appears that untreated serum used in growth media for culture cells contains plasminogen and plasmin which are known to proteolytically cleave t-PA (Wallen et al., supra). Adsorbent substrates such as Lysine-Sepharose chromatography have been shown to be effective in the removal of these proteins from serum (Wu et al., Exp. Cell Research 96:37-46 (1975); Quigley et al., J. Biol. Chem. Vol. 249, pg. 4306-4311 (1974)). Such depleted serum is capable of supporting the growth of tissue culture cells Wu et al., supra: Kaufman et al., Molec. Cellular Biology 5:1750-1759 (1985)).